From ancient times, cats have been favorably treated as a pet by humans. In modern Europe and America, they are called "Companion species" and now becoming a member of human society. On the other hand, cats have hitherto been used as an experimental animal in various fields including medicine, pharmacy, animal husbandry, veterinary, psychology etc. They are also used as a minimal disease cat in recent years in tests for determination of effect and safety of drugs, and hence, usefulness thereof for humans becoming greater and greater. In any case, it is earnestly desired to establish a method for more certain diagnosis, treatment and prevention of feline diseases, especially feline infectious diseases in order to keep cats in healthy conditions thereof.
There are many feline viral diseases, and among them, those caused by feline rhinotracheitis virus, feline parvovirus, feline infectious peritonitis, etc. are acute diseases having a very high lethality rate. Although vaccines for prevention of these diseases have been developed, only symptomatic therapy such as by antibiotics and sulfonamides which prevents secondary bacterial infections has been available for treating those cats infected and attacked with these diseases, and hence, the conventional methods for treating these diseases are still insufficient.
Hitherto, a hyperimmune serum and an immunoglobulin derived from serum have been utilized for treatment of these diseases and confirmed to be effective. However, with the popularity of the idea for kindly treatment of animals, feline serum materials have become hard to obtain, and hence, this treatment can not be used nowadays. Therefore, development of a monoclonal antibody capable of neutralizing the infected viruses in place of the conventional hyperimmune serum will greatly contribute to the treatment of these viral diseases.
As mentioned above, a monoclonal antibody having a neutralizing activity against viruses can be used as the alternative to the hyperimmune serum. Hitherto, basic techniques of preparing monoclonal antibodies have been established mainly for a mouse monoclonal antibody. Monoclonal antibodies produced by cells such as hybridomas can advantageously be obtained in a large amount and semipermanently and solve the problem of material insufficiency. However, the monoclonal antibody in this case should be a feline monoclonal antibody instead of the conventional mouse monoclonal antibody in order to eliminate side effects such as anaphylatic shock, serum disease, etc. caused by the use in cats of the mouse monoclonal antibody which acts as a heteroprotein to cats.
Methods for preparing such feline monoclonal antibody as a drug for treating the feline viral diseases include:
(1) a method using a cat-cat hybridoma; PA1 (2) a method using a feline lymphocyte transformed with some viral or chemical agent; PA1 (3) a method using a cat-mouse heterohybridoma; PA1 (4) a method using a cat-(cat-mouse) hybridoma derived from a cat-mouse heterohybridoma; and PA1 (5) a method by gene recombination techniques of a mouse (V)--cat (C) chimeric monoclonal antibody wherein a variable (V) region which binds to an antigen is derived from a mouse monoclonal antibody having neutralizing activity against viruses and a constant (C) region which is responsible for antigenicity, immunogenicity and physiological activity is derived from a feline monoclonal antibody.
However, none of the above methods have hitherto been reported to be effectively used.
In the method (1), a fusion efficiency is quite low and no appropriate myeloma strain is available. In case of the method (2), there are no appropriate virus corresponding to EB virus in case of human and no appropriate chemical agents. The methods (3) and (4) will have much difficulty (for example, a stability problem etc.) in obtaining the desired feline monoclonal antibody with high efficiency in view of the case of preparation of a human monoclonal antibody. Therefore, it is expected that the method (5) using the chimeric monoclonal antibody is the most realizable method among these five methods.
The chimeric monoclonal antibody is prepared by incorporating a plasmid vector containing a mouse (V)--cat (C) chimeric antibody gene into an animal host cell (e.g. mouse myeloma cell), expressing said gene in the host cell and collecting the monoclonal antibody from a supernatant of the culture, wherein said mouse (V)--cat (C) chimeric antibody gene is such that a V (variable) gene is cloned from a mouse-mouse hybridoma capable of producing a mouse monoclonal antibody as a source of a gene coding for a V region, a C (constant) gene is cloned from a feline cell such as a feline antibody-producing cell capable of producing a feline monoclonal antibody as a source of a gene coding for a C region and said V gene and said C gene are linked to each other. Several reports are found as to human chimeric antibodies (Japanese Patent First Publications Nos. 155132/1985 and 47500/1986).
As mentioned above, a gene coding for an amino acid sequence in a variable (V) region of an antibody molecule capable of binding to a desired antigen and a gene coding for an amino acid sequence in a constant (C) region of a feline immunoglobulin are required for preparing the feline chimeric antibody. The gene coding for the variable (V) region of the chimeric antibody is derived from a cell capable of producing a mouse monoclonal antibody having a neutralizing activity against the above mentioned various feline viruses and said cell can be prepared rather easily by the conventional mouse-mouse hybridoma producing procedure. However, the gene coding for the constant (C) region of the chimeric antibody, i.e. the gene coding for the constant (C) region of the feline immunoglobulin is still unknown in its structure and has never been cloned. Therefore, in order to prepare the feline chimeric antibody, it is inevitably required to find the gene coding for the amino acid sequence of the constant (C) region of the feline immunoglobulin.
In addition, although there is much difficulty in obtaining the monoclonal antibody showing a desired specificity in case of the methods (1) to (4), materials (cell strains) effective for preparing the chimeric antibody can effectively be provided in case of the method (5) since any cell which produces the feline immunoglobulin regardless of its specificity can preferably be employed as material for providing a gene coding for the C region of the feline immunoglobulin for preparing the chimeric antibody.